My LC Blog: The Detector

Next to the pump and separation column, the detector is another essential part of any HPLC system - without it there is no signal. Ideally this signal will provide a multitude of information. Not only do we learn that there is something eluting from the column, but if things go well and we have good separation, we will also gain knowledge on how many analytes of interest are in the sample, and the concentration of each single compound (quantitative analysis). In addition, some detectors will also provide supplementary data to identify the peaks (qualitative analysis), not only by retention time, but also spectral or mass data.

In HPLC we use a variety of detectors with different advantages, the most common by far are the so-called absorbance detectors. When light hits the compound, the latter absorbs light of a specific wavelength. The wavelength of light absorbed, or whether the analyte absorbs light at all, depends on the structure of the substance, it must contain at least one chromophore (see glossary below). Light in the ultraviolet to visible range is used for absorbance detection.

Quantitative analysis in HPLC using absorbance detectors is based on Lambert-Beer's Law. Simply put, it states that absorbance and concentration are linearly related, meaning that if you plot absorbance against concentration you will obtain a straight line, as shown in figure 1. By creating a so-called calibration curve with standards of known concentration, you can calculate concentrations anywhere on the curve within this linear range.

In an ultraviolet detector (UV) a deuterium (D2) lamp is used as a source for ultra-violet light (190 - 600 nm), while a UV/Vis detector also contains a tungsten lamp to include the visible wavelength range (> 400 nm).?The light emitted from the lamp is separated into a light beam of a certain wavelength. It then passes through the flow cell and enters the photodetector (photodiode). The electrical signal corresponding to the intensity of the emitted light is reduced when a compound that absorbs light at the given wavelength passes through the cell (figure 1). The higher the sample concentration, the higher the absorbance, the higher the peak in the chromatogram.

Figure 2 shows the schematic diagram of a photodiode array detector (PDA) or diode array detector (DAD). Light passes through the flow cell before being dispersed using a diffraction grating. It is then received by a diode array element that monitors the intensity of light at each wavelength.

As a multi-channel detector the PDA permits access to spectral data for several wavelengths simultaneously, which allows measurement of a continuous UV spectrum, resulting in a multi-wavelength chromatogram. As can be seen in figure 2, the detector provides not only a chromatogram that refers to time on the X axis and absorbance on the Y axis, as obtained with the UV detector, but also three-dimensional data showing the different wavelengths on the Z axis. Many compounds can be unequivocally identified using their UV-spectrum, which is why the PDA detector is very popular for HPLC method development. Follow this link for an application example: eL546 Qualitative Analysis of UV-Absorbents in Cosmetics Based on UV-Vis Spectrum (shimadzu.com)

UV and PDA detectors are the most common HPLC detectors, thanks to their reliability, ease of use, and universal response to chromophoric compounds. They are also non-destructive, meaning the sample leaves the detector unchanged, which is essential in case you want to collect the single compounds for further investigation. However, there are other options that I have summarized in table 1.

But first I think today we need another glossary:

• Spectral data: Data measured for specific wavelengths of the electromagnetic spectrum. In HPLC analysis with absorbance detection, this refers to chromatograms measured at different wavelengths, or the full UV spectrum of a compound. It can also be used to determine peak purity, which affects the absorbance behavior of a compound.
• UV-Spectrum: Plot of absorbance vs. wavelength for a specific compound. It can be used for compound identification and also to determine the optimum absorbance wavelength to acquire maximum detector sensitivity for the analyte of interest.
• Chromophore: A chromophore is a moiety in the chemical structure of a compound that absorbs UV or visible light. It possesses electrons that can absorb energy which converts them from their ground state (lowest state) to an excited state (high energy state).

Table 1: HPLC detector options (not a complete list)

Let me briefly describe the functionality of these typical LC detectors:

• Fluorescence detector (RF): Electrons in fluorescent molecules can be excited using a specific wavelength - the excitation wavelength - and then emit light of a different wavelength (emission wavelength). The intensity of this emitted light is monitored to quantify the analyte concentration. See also: Fluorescence Detection : SHIMADZU (Shimadzu Corporation)
• Refractive index detector (RID): The RID measures any change in the refractive index - meaning the diffraction of a light beam by the content of the flow cell - compared to a reference cell filled with only mobile phase. See also: Refractive Index Detection (RID) : SHIMADZU (Shimadzu Corporation)
• Conductivity detector (CDD): ?The CDD detects electrical conductivity of ions. Voltage is applied to a pair of plates, and if ions are present, a current flows. If there are ions in the mobile phase as it passes through the detector flow-cell, a signal is produced that is proportional to the ion concentration.
• Evaporative light scattering detector (ELSD): Detects the light scattered by particles remaining after evaporation of the mobile phase.

You may have noticed that there is no Mass Spectrometer (MS) mentioned in the list of detectors, why is that? Well, first of all an MS is NOT simply an LC detector, at least that’s what my colleagues tell me. And secondly – it’s really not my area of expertise. So instead of copying some expert knowledge from a textbook – which anyone could do – I’m going to ask one of my colleagues with actual experience in the field, to be my first guest writer. And since it was pointed out to me that the MS is vital for peak tracking in method development, we’ll do that in the next month, before starting with the topic of HPLC method development.